TECHNICAL USER GUIDE. Mimetic Blue SA HL P6XL. (Product Code: 3125) TECHNICAL USER GUIDE

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1 TECHNICAL USER GUIDE TECHNICAL USER GUIDE Mimetic Blue SA HL P6XL (Product Code: 3125)

2 Introduction Mimetic Blue SA HL ( high ligand ) P6XL affinity chromatography adsorbent is developed and manufactured exclusively by ProMetic BioSciences Ltd (PBL). It comprises Mimetic Blue ligand covalently attached to a near monodisperse 6% crosslinked agarose bead (PuraBead 6XL) and is immobilised using a defined and highly stable spacer-arm linkage which promotes optimal interaction with the target protein. Properties of Mimetic Blue SA HL P6XL Ligand: Adsorbent appearance: Mean particle size: Matrix: Binding capacity: Recommended packing conditions: Recommended operational flow rates: Operating ph: Synthetic anthraquinone Blue 100 μm ± 10 μm PuraBead 6XL (6% cross-linked near monodisperse agarose) Up to 60 mg HSA/mL of adsorbent Packing pressure - 2 to 3 bar Packing solution M NaCl solution Up to 500 cm/hr with column diameter less than 3 cm Up to 200 cm/hr with column diameter greater than 5 cm ph 2 to ph 14 (intermittent) ph stability: Long term (3 months) ph 3 to ph 13 Chemical stability: All commonly used aqueous buffers and co-solvents Cleaning/sanitization: M NaOH, 25 C Sterilization: Storage conditions: Autoclavable in 0.1 M saline at 121 C for 30 minutes 2-30 C, 20% ethanol/80% 0.1 M NaCl (v/v) 2

3 Column Packing Mimetic Blue SA HL P6XL is supplied in 20% ethanol/80% 0.1 M NaCl (v/v) solution. Before commencing the column pack, consult the relevant manufacturer s instructions for the selected column. The method below describes the packing of Mimetic Blue SA HL P6XL into axial columns: 1. Decant off the shipping preservative and prepare a 50% slurry of the adsorbent with 0.1 M NaCl solution. 2. Assemble the column and remove air from the dead spaces by flushing the end piece and adaptor with packing solution (0.1 M NaCl solution) then close the column outlet. 3. Allow all materials to equilibrate to the temperature at which the chromatography process is to be performed. 4. If required to obtain a fixed bed height (i.e. for larger column packs), it is recommended to accurately determine the slurry percentage. For example; weigh a sample of the complete slurry, drain away the preservative and re-weigh the adsorbent. The final weight over the total weight will determine the slurry percentage. 5. Carefully pour the adsorbent slurry into the column in a single continuous step (use a reservoir if required). Pouring the adsorbent down the side of the column helps to prevent air becoming trapped within the adsorbent bed. 6. Allow the adsorbent to settle in the column just enough to leave a dead volume of packing solution above the adsorbent. 7. Attach the (open) top adaptor to the top of the column and adjust the adaptor to just above the bed, tighten the adaptor and attach to the workstation. Open the column outlet and apply the desired flow to the bed. The recommended packing conditions (to obtain a uniform pack) is at a constant pressure up to but not exceeding 3 bar (~45 psi). 8. Once the adsorbent has packed (after ~2 CV), measure and mark the bed height under packing flow, close the column outlet and stop the liquid flow through the bed. 9. Lower the top adaptor by loosening the top adaptor seal (the top adaptor must allow free flow from the workstation either by loosening the top adaptor connection or if present switching the top valve to waste) to the position of the marked bed height (do not push the top adaptor further into the adsorbent bed). Note: once the flow is paused the bed may relax and rise. 10. Re-tighten the top adaptor (if loosened) and attach back to the workstation (or switch valve back in-line). Open the bottom outlet and apply the packing flow to the column again for 1 CV. If a space is formed between the top of the bed and the adaptor repeat the steps above. If no space forms the column is packed and ready to use. 3

4 Operating Instructions Performance Testing of the Packed Column To check the quality of the column pack, the height equivalent to a theoretical plate (HETP) and peak asymmetry need to be determined. This can be achieved by quantifying the geometry of an isocratic peak, which can be produced by injecting a marker detectable by spectrophotometry or conductivity. The recommended conditions are to inject a 2% CV of a solution of 2% acetone or 2.0 M NaCl (measurable by UV 280 nm or conductivity respectively) at a flow rate of 100 cm/hr, using saline or equilibration buffer as the mobile phase. Recommended Chromatography Buffers for Using Mimetic Blue SA HL P6XL Note: The following recommendations are not prescriptive and thorough investigation of these parameters at small-scale should be conducted to reveal the level of flexibility that can be tolerated with the chromatography adsorbent, buffer and protein combination selected. Mimetic Blue SA HL P6XL PuraPlate (code: 2231) or 1 ml Column Kits (code: ) are also available for high throughput process optimization and scouting experiments. Sample treatment: Clarify the feedstock/protein solution using an appropriate filter and adjust the ph and or conductivity of the solution if required. Equilibration buffer: 25 mm sodium phosphate buffer at ph 6.0, however other buffers are acceptable with a recommended ph range between ph 4.0 and ph 8.0. Wash buffer: If a wash step is required to remove any loosely bound non-target material, initially a moderate amount of salt (up to 0.2 M NaCl) can be added to the equilibration buffer to remove non-specifically bound proteins. Alternatively, additional washes of buffers with increasing ph can also have the same desired effect. Elution buffer(s): Albumin and albumin related proteins are selectively eluted from Mimetic Blue SA HL P6XL using up to 60 mm sodium octanoate (caprylate) present in the equilibration buffer. Note: Increasing the concentration of salt (up to 2.0 M NaCl) will non-selectively promote elution from the adsorbent. Cleaning-in-Place (CIP) and Sanitization: Removal of any residual adsorbed material including micro-organisms, viruses and 4

5 endotoxins can be achieved by washing the column with 0.5 to 1.0 M NaOH. A contact time of 1 hour will normally suffice to ensure destruction of viable organisms, although up to 5 hours contact time may be required. No less than 5 column volumes are recommended. Once cleaning with NaOH is complete, wash with at least 5 bed volumes of equilibration buffer until the ph and conductivity of the column eluate is equal to that of the buffer entering the column. Complete this process prior to further use or storage in the storage buffer. Storage: Mimetic Blue SA HL P6XL should be stored in 20% ethanol/80% 0.1 M NaCl (v/v) at 2-30 C. Note: After long term storage ( 3 months), it is recommended to wash the adsorbent with 5 bed volumes of 30% isopropanol/0.2 M NaOH solution at a linear flow rate equivalent to 50% of the operational flow rate. Once completed re-equilibrate the adsorbent prior to use. If you have any questions please contact us on Recommended Chromatography Conditions for Using Mimetic Blue SA HL P6XL The following method is recommended (as a starting point). An initial flow rate of 100 cm/hr for the column chromatography steps is recommended. Subsequent increases/decreases in the flow rate can be investigated to improve binding capacity/resolution or decrease processing times. 1. Equilibrate the column with 3 CV of equilibration buffer or until the ph/conductivity is at baseline (Note: ensure the equilibration buffer of the column is comparable to the protein feedstock). 2. Apply the protein feedstock onto the equilibrated column. A residence time of 3 minutes (or greater) is recommended. 3. Remove any non-bound material in the column with 5 CV of wash buffer, or until the UV trace returns to baseline. 4. Elute the bound protein using 25 mm sodium phosphate, 30 mm sodium octanoate ph 6.0 with up to 5 CV of elution buffer. 5. If a CIP is required, pass 5 CV of 0.5 M NaOH through the column. 6. Re-equilibrate column with 5 CV of equilibration buffer (until the ph of the eluent is comparable to the equilibration buffer entering the column). 7. For later date use, place the column into the storage solution and store at 2-30 C. 5

6 Order information Gel Slurry Code Description Contents Mimetic Blue SA HL P6XL 25 ml Mimetic Blue SA HL P6XL 100 ml Mimetic Blue SA HL P6XL 500 ml Mimetic Blue SA HL P6XL 1,000 ml We also offer a range of larger pack sizes for supply of bulk resins into cgmp development and manufacturing scale processes. For more information on this or any other supply related matters please do not hesitate to contact us on sales-pbl@prometic.com Column Format (5 x 1 ml) Code Description Mimetic Blue SA HL P6XL 1 ml Column Kit 96 Well Format (96 x 0.25 ml) Code Description 2231 Mimetic Blue SA HL P6XL PuraPlate Mimetic Blue ELISA Kit Code Description 2090 ELISA Kit (Mimetic Blue 1/Blue SA) 6

7 Sales & Technical Support NORTH AMERICA ProMetic BioSciences Ltd 1330 Piccard Drive Suite 201 Rockville MD 20850, USA Tel: Fax: EUROPE/REST of the WORLD ProMetic BioSciences Ltd The Freeport Ballasalla Isle of Man IM9 2AP, British Isles Tel: +44 (0) Fax: +44 (0) SALES TECHNICAL SUPPORT- WEBSITE - ProMetic BioSciences Ltd and the PBL logo are trademarks of ProMetic BioSciences Ltd. PuraBead and Mimetic Blue are registered with the US Patent & Trademark Office. Issue ProMetic BioSciences Ltd